Integrative and Comparative Biology
Integrative and Comparative Biology, volume 53, number 6, pp. 1002–1013
doi:10.1093/icb/ict047
Society for Integrative and Comparative Biology
SYMPOSIUM
Coping with Uncertainty: Nutrient Deficiencies Motivate Insect
Migration at a Cost to Immunity
Robert B. Srygley1,* and Patrick D. Lorch†
*Northern Plains Agricultural Research Lab, USDA-Agricultural Research Service, 1500 N. Central Avenue, Sidney,
MT 59270, USA; †Biological Sciences Department, Kent State University, Kent, OH 44242, USA
From the symposium ‘‘Coping with Uncertainty: Integrating Physiology, Behavior, and Evolutionary Ecology in a
Changing World’’ presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7,
2013 at San Francisco, California.
1
E-mail: [email protected]
Synopsis Migration often is associated with movement away from areas with depleted nutrients or other resources, and
yet migration itself is energetically demanding. Migrating Mormon crickets Anabrus simplex (Orthoptera: Tettigoniidae)
lack nutrients, and supplementation of deficient nutrients slows migratory movements and enhances specific aspects of
their immune systems. Migrants deficient in proteins have less spontaneous phenoloxidase (PO) activity, whereas those
deficient in carbohydrates have lower lysozyme-like anti-bacterial titers with a proposed compromise between migratory
and anti-bacterial activities. To investigate the relationship between diet, movement, and immunity further, we removed
Mormon crickets from a migratory band and offered each cricket one of five diets: high protein, high carbohydrate, equal
weight of proteins and carbohydrates (P þ C), vitamins only, or water only for 1 h. We then attached a radio, returned
each to the migratory band, and recaptured them 18–24 h later. Mormon crickets fed protein moved the furthest, those
with only water or only vitamins moved less, and those fed carbohydrates or P þ C moved the least. Standard intake trials
also indicated that the Mormon crickets were deficient in carbohydrates. Consistent with a previous study, lysozyme-like
anti-bacterial activity was greatest in those fed carbohydrates, and there was no difference between those fed water,
protein, or P þ C. Crickets were removed from the same migratory band and fed one of four diets: high P, high C, P þ C,
or vitamins only, for 1 h. Then the crickets were held in captivity with water only for 4 or 24 h before blood was drawn.
Immunity measures did not differ between times of draw. Diet treatments had no effect on anti-bacterial activity of
captive Mormon crickets, whereas total PO was greater in those fed protein. These results support the hypothesis of a
direct compromise between migratory and anti-bacterial activities, whereas PO is compromised by low protein independent of migratory activities. We discuss the potential effects of climate on nutritional deficits and susceptibility to
different pathogens.
Introduction
Balancing dietary intake to optimize allocation to
growth, maintenance, and reproduction is a challenge for organisms in a changing environment
(Rapport 1980; Simpson et al. 2004). Scarcity of nutrients or other resources may result from changes in
temperature, humidity, or other abiotic factors affecting primary productivity, or from interspecific
or intraspecific competition, such as local crowding
(Farrow 1990; Despeland et al. 2000). Scarcity in
local nutrients or other resources can motivate animals to migrate. As a result of local scarcities, some
or all members of the population move to sites
where resources may be more abundant. However,
moving long distances is also energetically demanding. If changes in primary productivity or consumer
density can be predicted, then animals may prepare
for their migratory behavior by depositing fat before
food becomes scarce. However, changes in primary
productivity and consumer density often occur without warning, and particularly when critical nutrients
are scarce initially, animals find themselves poorly
nourished (Mattson and Haack 1987). Animals in
nutrient-poor habitats may redirect resources from
other life-history traits, such as reproduction
(Johnson 1969; Zera and Harshman 2001) or
Advanced Access publication May 13, 2013
Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology 2013. This work is written by US
Government employees and is in the public domain in the US.
Diet deficiency, migration and immunity
immunity (Srygley et al. 2009; Srygley and Lorch
2011), to fuel migration.
Migrating bands of Mormon ‘‘crickets’’ (actually
katydids), common during the current outbreak in
the U.S. Great Basin, are an excellent model organism for understanding mass migrations of grasshoppers, locusts, and other insects. They are also useful
for understanding interactions between diet, migration, and immunity. Mormon crickets in some bands
seek proteins over carbohydrates for their diet
(Simpson et al. 2006; Srygley et al. 2009), whereas
in other bands they prefer carbohydrates over
proteins (Srygley and Lorch 2011). In locusts, preferences for particular nutrients depend on deficiencies due to past diets (Raubenheimer and Simpson
2003). Although studies of dietary effects on immunity are rare (Schmid-Hempel 2005), limitation of
nutrients appears to be detrimental to immune function (Moret and Schmid-Hempel 2000; GonzálezTokman et al. 2011). Mormon crickets with different
dietary needs also contrast strongly in their immune
systems; protein seekers have less phenoloxidase
(PO) enzyme circulating in their blood, whereas carbohydrate seekers have less anti-bacterial enzyme
activity (Srygley et al. 2009; Srygley and Lorch
2011). Only two other studies have addressed the
role of nutrition and immunity in Orthoptera
(Jacot et al. 2005; Adamo et al. 2010), and only a
few have investigated it in other insect orders
(Coleoptera: Rantala et al. 2003; Lepidoptera: Ojala
et al. 2005; Lee et al. 2006; Klemola et al. 2007; Lee
et al. 2008; Povey et al. 2009). Parental diet has
transgenerational effects in Plodia moths, with poor
diets for either or both parents causing decreased PO
titers and fewer circulating hemocytes in offspring
(Triggs and Knell 2012).
In Mormon crickets, we hypothesize that a direct
conflict exists between migratory and anti-bacterial
activities and is most evident when dietary carbohydrates are limited and fats are shuttled to the muscles
as fuel for migration (Srygley and Lorch 2011). We
focus on lysozyme-like activity against Gram-positive
bacteria for our measure of antibacterial activity in
Mormon crickets because, unlike other insects, antimicrobial peptides (AMPs) are little known in
Orthoptera. As far as we are aware, locustin is the
only AMP identified to date (Swiss-Prot: P83428.1;
characterized from migratory locust hemocytes with
activity against Gram positive Micrococcus luteus). No
AMPs have been found in other orthopterans.
Adamo (2004) suggested that activity of the hemolymph of the cricket Gryllus against Gram negative
bacteria indicates it has more than just lysozyme-like
enzymes. However, this conclusion comes from the
1003
assumption that lysozyme-like enzymes are not
thought to be active against Gram negative bacteria.
In Lepidoptera, some lysozymes are active both
against Gram-positive and Gram-negative bacteria
(Gandhe et al. 2007; Wang et al. 2009). Hoffmann
et al. (1996) suggested that the orthopterans might
also rely on lysozyme-like enzymes for defense
against both types of bacteria, but more work
needs to be done on AMPs in Orthoptera.
In contrast, we hypothesize that PO is compromised by poor protein nutrition independent of
migratory activities (Srygley et al. 2009). PO is a
key enzyme in the generalized immune response of
insects to wounding and invasion. PO catabolizes
tyrosine to produce toxic quinones that polymerize
to form melanin (for a recent review, see GonzálezSantoyo and Córdoba-Aguilar 2012). The PO cascade
is an important part of cellular encapsulation of foreign bodies because melanization hardens the cell
mass and suffocates intruders (Kanost and Gorman
2008).
Seeking to compare the effects of protein and carbohydrate diets with that of diets lacking macronutrients, we offered a range of diets (with and without
protein, carbohydrates, and essential vitamins, minerals, and oils) to Mormon crickets before releasing
them back into a migrating band. For Mormon
crickets with a carbohydrate deficiency, we predicted
results consistent with those from the previous year:
migratory velocity would decrease and lysozyme-like,
anti-bacterial enzymatic activity would increase following consumption of a diet rich in carbohydrates
compared with a diet rich in proteins. We also predicted that the two sets of controls (vitamins only
versus wildtypes) would be similar in migratory velocity and enzymatic immunity. We predicted that
(1) those fed a diet rich in carbohydrates would
have a migratory velocity less than that of the two
sets of controls, (2) anti-bacterial activity would be
greater than for the two sets of controls, and (3)
these activities of Mormon crickets given a diet
rich in protein would not differ from the controls.
Finally, we predicted that (4) a P þ C diet would be
intermediate between those fed either P or C. In
other words, those fed a P þ C diet would probably
not differ substantially from the controls.
In addition, we investigated whether dietary effects
on enzymatic immunity of captive insects reduced
the effects of migration. Because we hypothesized
that locomotion compromises anti-bacterial activity,
we predicted that anti-bacterial activity would be
independent of diet in captivity. Our prior studies
differed in the time between dietary supplementation
and sampling of hemolymph, and so we also
1004
explored whether this lag affects the PO and antibacterial enzymatic activities. Finally, we discuss possible consequences of nutritional deficits driven by
abiotic factors for infections by fungi and bacteria.
Materials and methods
Mormon cricket bands and the study site
Mormon crickets, Anabrus simplex (Orthoptera:
Tettigoniidae), are univoltine katydids that hatch in
the spring from eggs laid singly in the soil. From the
fourth instar onward they can begin to migrate,
while completing their seven nymphal instars and
molting to adults. As teneral adults, they gain additional weight and become reproductively mature in
approximately 6–8 days in males and 10–12 days in
females. Very little is known about the ecological
mechanisms underlying outbreaks, aggregation, or
migration. Outbreaks typically originate on rangeland and are characterized by spectacular migratory
bands that can be over 10 km long, several kilometers
wide, contain dozens of insects per square meter, and
travel up to 2 km per day (Cowan 1929; Wakeland
1959; MacVean 1987; Lorch and Gwynne 2000;
Gwynne 2001; Lorch et al. 2005). The current outbreak began in 1998 and is one of the most severe on
record, with 5 million hectares in Nevada alone estimated to have been infested in 2004 (Knight 2007),
which is six times the area recorded in 1939 for
Nevada during another historic outbreak (Cowan
1990).
Between June 30 and July 7, 2009, we studied a
band of pre-reproductive adult Mormon crickets in
Independence Valley (latitude 418 130 1200 N, longitude 1168 20 3500 W, elevation: 1940 m) of the
Independence Range in Elko County approximately
50 km northwest of Elko, Nevada. This site was only
3 km from the band studied at Eagle Mountain in
2008 and described by Srygley and Lorch (2011). The
density of adult Mormon crickets in the band at
Independence Valley ranged from 10.3 to 14.0 m2,
and they were heading 708 from geographic North
(R. B. Srygley, unpublished data). The floor of the
valley is dominated by sagebrush (Artemisia tridentata), typical vegetation for high Great Basin desert
scrubland. Mormon crickets are omnivorous; they
eat broad-leafed plants, invertebrates, and fungi,
scavenge on dead vertebrates and road kills, and cannibalize other Mormon crickets (Ueckert and Hansen
1970).
Intake trials
We characterized the dietary intake of field-collected
Mormon crickets following the methods of Simpson
R. B. Srygley and P. D. Lorch
et al. (2006). We prepared a 42% protein diet consisting of a 3:1:1 mix of casein, peptone, and albumen and a 42% carbohydrate diet consisting of equal
parts of sucrose and dextrin. Both diets contained
54% cellulose and 1.8% Wesson’s salt mixture and
2.2% vitamins, linoleic acid, and cholesterol. In a
free-choice experiment, four male and four female
Mormon crickets were collected from the migratory
band on June 30, housed individually with free
access to water and 0.5 g of each diet for 24 h.
After 24 h, the diets were removed and replaced
with fresh diet, which remained with the insects for
an additional 24 h. As a replicate, five males and
three females were collected on June 30 from a
second band that we did not radiotrack and given
the same choice of diet. This band was 7 km east of
the band used for release-recapture and for the laboratory experiments described below. The dry masses
consumed from each diet is a measure of the relative
intake of carbohydrates:protein over the first and
second 24-h period. The diet provided during the
second 24-h period is closer to the ideal intake for
the band. A vector drawn from the mean intake
during the first 24-h period and that for the
second 24-h period indicates the dietary needs of
members drawn from the migrating band relative
to the same insects with free choice of diet. A migrating band is deficient in protein (or carbohydrate)
when its members prefer to feed on proteins (or
carbohydrates) when first captured and then switch
to a more even ratio of proteins and carbohydrates
(Simpson et al. 2006).
Manipulation of diet, radio-tracking, and collection
of hemolymph
We captured 25 males and 25 females on July 3 and
20 males and 20 females crickets on July 6, 2009.
Each was held in a 1-l translucent container and
given one of the five diets: a 42% protein diet, a
42% carbohydrate diet (see above), a diet composed
of equal parts protein and carbohydrate (21% for
each constituent), a control diet lacking macronutrients (this we call the O diet: 96% cellulose, 1.8%
Wesson’s salt mixture, and 2.2% vitamins, linoleic
acid, and cholesterol), and another control group
that did not receive any food (these we refer to as
wildtypes). The insects were shaded beneath a tarpaulin and allowed to feed from their diet for 1 h
between 15:30 and 16:30 h. In order to measure consumption, the food was weighed to the nearest milligram on an Ohaus field-portable microbalance
(model AV53) before and after presentation to the
crickets.
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Diet deficiency, migration and immunity
In order to follow each Mormon cricket’s migratory path, a 0.4 g radio-transmitter (Biotrack, Ltd.,
UK) was glued to the pronotum. The added mass
of the radio did not affect locomotion in the laboratory (Lorch et al. 2005). Sex, treatment, and the
unique radiofrequency that identified each insect
were recorded. Using a Trimble GPS datalogger to
record location and time for each cricket, we released
the insects back into the band with each cricket initially separated from other marked individuals by ca.
8 m on a linear transect perpendicular to the general
direction of movement of the band at that time. On
July 3, crickets were released into the band between
18:30 and 18:55 h and retrieved the following day
between 11:00 and 17:00 h (PDT) for the first
group (six were not recaptured until July 5 between
11:00 and 17:00). Two from this first group were
excluded because they were either predated or lost
their radio within 7 m of release. On July 6, crickets
were returned to the band between 18:15 and 18:35
and recovered the following day between 10:30 and
15:15 (PDT). One from this second group was predated within 12 m of release. The location (1 m)
and time of recapture were recorded, and each
cricket was placed in a 50-ml Corning plastic centrifuge tube. Velocity (mm s1) was calculated as the
straight-line distance between release and recapture
over time.
Returning to our field laboratory in Taylor
Canyon, Nevada, in the early afternoon, we measured body mass of each cricket to the nearest milligram with the Ohaus microbalance. We drew
hemolymph by puncturing the arthrodial membrane
at the base of each insect’s hind leg with a 26-gauge
hypodermic needle. In Orthoptera, prophenoloxidase
(proPO) is primarily held in circulating hemocytes
until wounding or infection (Kanost and Gorman
2008). Hence lysis of hemocytes at the wound may
cause local elevation of proPO and initiation of the
proPO cascade to yield spontaneously-active PO.
Puncturing again, if necessary, 15 ml of hemolymph
was collected into a capillary tube. The hemolymph
was diluted 1:50 with phosphate buffered saline
(PBS) solution to be used in assays of spontaneous
PO and proPO enzymatic activity and total hemolymph protein. An additional capillary tube with
10 ml of hemolymph diluted 1:4 with PBS was collected for assay of anti-bacterial activity. We immediately froze the hemolymph samples at 188C in a
field-portable Engel freezer (model MT45F-U1).
We captured a second set of 48 Mormon crickets
from the same band and held each in a 1-l translucent container. Each was given one of the four diets:
P, C, P þ C, or O. The insects were allowed to feed
from their diet for 1 h between 13:20 and 14:20. In
order to measure consumption, diets were weighed
to the nearest milligram on the microbalance before
and after presentation to the crickets. These were
held in captivity in the field laboratory on natural
photoperiod for an additional 4 or 24 h when hemolymph was drawn and stored, as described for the
released-recaptured crickets above.
Immunocompetence assays
We followed the protocols detailed by Srygley and
Lorch (2011). Briefly, samples of thawed hemolymph
diluted in PBS were centrifuged and activated with
10 mM dopamine solution to measure spontaneous
PO activity. The plate was loaded into a temperature-controlled Biotek microplate reader (258C), and
absorbance at 492 nm was read between 5 and
15 min. If absorbance of the sample was linearly related with time, we calculated mean V (change in
absorbance min1). One unit PO activity per milliliter of hemolymph is defined as the amount of
enzyme resulting in a 0.001 increase in absorbance.
To measure total combined activity of PO and
proPO, we adapted the protocol of Goldsworthy
et al. (2002). We dissolved 1 mg alpha-chymotrypsin
from bovine pancreas (Sigma) in 1 ml PBS, combined an equal volume of this solution with centrifuged hemolymph in PBS (1:50), and incubated for
30 min. In the plate wells, we added 5 ml of the incubated solution to 195 ml 10 mM dopamine. As
above, mean V was calculated from plate readings
between 5 and 15 min to measure total PO activity
in units ml1 of hemolymph.
To measure lysozyme-like antibacterial activity, a
turbidimetric method was used. Ten microliters of
thawed and PBS-diluted hemolymph (1:4) was
added to a well with 140 ml of Gram-positive cells
of the bacterium Micrococcus lysodeikticus (Worthington) suspended in PBS (0.5 mg/ml). Clearing of
the well was compared with a serial dilution of
egg-white lysozyme (Sigma) added to the bacterial
suspension. The plate was loaded into a temperature-controlled Biotek microplate reader (258C) and
absorbance at 450 nm read between 10 and 30 min. If
the sample absorbance was linearly related with time,
we calculated mean V. When activity of the sample
fell below 6.5 mg ml1, the sample was excluded because the standards showed that such data were
unreliable.
Statistical analyses
To analyze the effects on marching velocity, which
did not differ from a normal distribution (P ¼ 0.66),
1006
we conducted a forward stepwise regression analysis
using JMP 6.0.2 (SAS Inc.) with velocity as the dependent variable, body mass as the covariate, sex,
day of release, and diet as independent factors. The
analysis first divided the five diet treatments to make
two group levels with the greatest separation between
means of the responses, and then those two groups
were further divided to maximize the separation
within each group, continuing hierarchically until
all levels fell into four terms. All two-way and
three-way interactions were evaluated. We selected
the model that minimized the Akaike Information
Criterion.
When we analyzed immunity measures of released-recaptured Mormon crickets to see how they
were affected by sex, diet, and body mass, not all
immunity assays were conducted on all insects, and
so we ran analysis of covariance (ANCOVA) on each
measure of immunity separately. Anti-bacterial activity was log10 transformed to normalize this dependent variable (P ¼ 0.41), whereas total PO did not
differ from a normal distribution (P ¼ 0.50). In
building our models, body mass was entered as a
covariate and sex and diet were independent factors
with all interactions evaluated. Interactions that were
not significant were removed from the model. For
significant factors, multiple comparisons of the least
squares means of the dependent variable were conducted post hoc using Student’s t-tests.
Because the distribution of spontaneous PO activity in released-recaptured Mormon crickets was bimodal, we used the non-parametric Kruskall–Wallis
test to evaluate differences among treatments within
each sex.
We used ANCOVA to evaluate effects of body
mass, lag-time, sex, and diet on anti-bacterial activity, total PO, and spontaneous PO activities of captive insects. Anti-bacterial activity and total PO were
log10 transformed to normalize their distributions.
We evaluated three-way interactions, which were
removed from the model if not significant. Then
we evaluated two-way interactions, which also were
removed from the model if not significant before
evaluating the main effects of the factors. Multiple
comparisons of the least squares means were conducted as stated above.
Results
Intake trials
Mormon crickets from the band in Independence
Valley ate more carbohydrate than protein with
about 1.5 times the consumption of C to P on the
first day, followed by a more even consumption of C
R. B. Srygley and P. D. Lorch
and P on the second (approximately 1.2:1 C:P,
Fig. 1). The disparity in consumption of C and P
on the first day was not as great as for the band
studied the previous year, which consumed a C:P
ratio as great as 5:1 on the first day of the experiment. Convergence of intake vectors from both years
(Fig. 1) suggests an intake target of approximately
1.3:1. Hence, the Mormon crickets from Independence Valley showed compensatory feeding behaviors
consistent with a deficiency of carbohydrates in their
natural diets (Simpson and Abisgold 1985).
Diet and migratory movement
Only the first hierarchical split in diet treatment was
selected to best explain migratory velocity. The two
diet levels that significantly separated the mean migratory velocities were C and P þ C combined, contrasted with P, O, and W combined (Fig. 2, Table 1).
The estimated mean velocity for C and P þ C combined (5.58 mm s1) was slower than that for the
other diet treatments combined (6.84 mm s1).
Diet and immunity in released and recaptured
Mormon crickets
Log lysozyme activity increased significantly with
body mass, and diet was also a significant factor
(Fig. 3, Table 2). As predicted, Mormon crickets
fed a C diet had greater anti-bacterial activity than
did those fed a P diet. Also as predicted, the two
controls were not significantly different in anti-bacterial activity and their means were intermediate between those fed C and those fed P. As predicted,
anti-bacterial activity of those fed P did not differ
significantly from either set of controls. However,
those fed a C diet were not significantly different
from those fed vitamins only, but did differ significantly from the wildtypes. Finally, Mormon crickets
fed a diet of both P and C were not significantly
different from those fed P alone, or from the wildtype controls.
Total PO (proPO þ spontaneous PO) increased
significantly with body mass but did not vary significantly with sex or diet (Fig. 4, Table 3). Within each
sex, spontaneous PO activity was not significantly
related to body size (n ¼ 36: F ¼ 0.11, P ¼ 0.74;
n ¼ 33 males: F ¼ 3.29, P ¼ 0.08) nor did it differ
significantly between dietary treatments (Fig. 5;
Kruskal–Wallis tests: females: 2 ¼ 5.27, d.f. ¼ 4,
P ¼ 0.26; males: 2 ¼ 1.03, d.f. ¼ 4, P ¼ 0.90).
Diet and immunity in captive Mormon crickets
Mass, sex, diet, and draw time did not have significant effects on log anti-bacterial activity of captive
1007
Diet deficiency, migration and immunity
Fig. 1 Results of intake trials of Mormon cricket bands near
Tuscarora Nevada showing vectors drawn from carbohydrate:protein dietary intake on day 1 (diamonds) to that on day 2
(squares). The bold arrow indicates the band whose members
were released and recaptured, and the vector for a second band
from Independence Valley measured simultaneously is also
shown. For comparison, intake vectors of two replicates from a
band at Eagle Mountain in the previous year (open symbols) is
adapted from Srygley and Lorch (2011). The dashed line indicates
consumption of an even ratio of carbohydrates to proteins.
insects (P ¼ 0.81, 0.60, 0.12, and 0.49, respectively,
Fig. 3; two-way and three-way interactions were
not significant). On average, log-transformed antibacterial activity was 3.215 0.018 (mean S.E.) at
4 h and 3.205 0.017 at 24 h. Log total PO activity
increased significantly with mass and was significantly affected by diet, but not by sex nor draw
time (Table 4, two-way and three-way interactions
were not significant). On average, log-transformed
total PO activity was 4.092 0.025 (mean S.E.) at
4 h and 4.045 0.022 at 24 h. Multiple comparison
of the mass-adjusted means indicated that animals
on P and P þ C diets had significantly higher total
PO activity than did those fed vitamins only (Fig. 4).
P, C, and P þ C were not significantly different, and
C and vitamins-only were not significantly different.
Spontaneous PO activity was significantly greater in
females than in males, but it was not significantly
affected by mass, draw time, or by diet (Table 5;
two-way and three-way interactions were not significant). Circulating titers of PO averaged 663 39
units/ml hemolymph in blood drawn at 4 h and
601 45 units/ml hemolymph at 24 h.
Discussion
Lacking carbohydrates in their diet, the Mormon
crickets we studied probably were migrating in
search of scarce macronutrients. Mormon crickets
that were removed from the band ate more carbohydrates than protein; and those that ate protein
Fig. 2 Least-squares means of migratory velocity (S.E.) of Mormon crickets covaried with body mass were dependent on diet
(P: protein, C: carbohydrate, PC: protein þ carbohydrate, O: vitamins þ salt control, W: unfed wildtype).
1008
R. B. Srygley and P. D. Lorch
moved more quickly after returning to the band than
did those that fed on carbohydrates. Those that were
fed vitamins only as well as the unfed wildtypes
moved like those fed only carbohydrates, which suggests that those fed a protein diet moved more.
Protein-fed Mormon crickets behaved as if they
had found an unsuitable patch in which to forage
and consequently migrated faster than did those
fed carbohydrates. Those fed equal parts of carbohydrates and proteins behaved like those fed carbohydrates alone, suggesting that it was an insufficiency
of protein in their diet that spurred them on. We
conclude that the Mormon crickets were migrating
through an environment relatively abundant in protein and scarce in carbohydrates.
In contrast to dietary effects on migratory activity,
lysozyme-like anti-bacterial activity of Mormon
crickets that were fed carbohydrates was greater
Table 1 Model that best explained migratory velocity of Mormon
crickets following stepwise regression on date of release, dietary
treatments, gender, size, and all two-way and three-way
interactions
Factor
SS
MS
d.f.
F
P
Diet (P&W&O-C&PC)
32.9
32.9
1
4.25
0.042
Error
650.1
7.7
84
than in those that were fed proteins. Anti-bacterial
activity of those that were fed vitamins only and the
unfed wildtypes were not significantly greater than in
those fed protein alone, which suggests that those fed
a carbohydrate diet showed an increase in anti-bacterial activity. Those fed equal parts carbohydrates
and proteins had enzyme concentrations like those
fed protein alone, suggesting that an insufficiency of
carbohydrate in their diet reduced their anti-bacterial
activity. These results are consistent with the hypothesized trade-off between locomotion and immune
activity mediated by apolipophorin III, which has
roles in both corporal functions (Weisner et al.
1997; Halwani and Dunphy 1999; Weers and Ryan
2006; Adamo et al. 2008, 2010; Srygley and Lorch
2011). When sugar concentrations in the hemolymph
are low, apolipophorin III associates reversibly with
high-density proteins that transport lipids from the
fat body. When sugar concentrations are high, lipidfree apolipophorin III increases anti-bacterial lytic
activity (Weisner et al. 1997; Halwani and Dunphy
1999).
In further support of a trade-off between locomotion and anti-bacterial activity, diet did not have a
significant effect on anti-bacterial activity of captive
Mormon crickets. However, we predicted that in the
absence of migratory activity, anti-bacterial activity
would increase for all diet treatments and approach
Fig. 3 Least-squares means of antibacterial activity (S.E.) covaried with body mass were dependent on dietary treatment in the
release-recapture experiment (open bars), whereas the effect of dietary treatment was not significant in the laboratory experiment
(gray bars; see legend of Fig. 2 for abbreviations). Different letters above the columns indicate significant differences in multiple
comparisons of the means (P50.05) within each experiment. For comparison with the field results, the dashed line indicates the mean
for the Mormon crickets in the laboratory experiment.
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Diet deficiency, migration and immunity
that of the Mormon crickets fed carbohydrates and
released back into the band. However, we found that
the average for captive animals was well below that
for animals fed carbohydrates and was nearer to
values for those fed P or P þ C and released back
into the band (Fig. 3). This suggests that apolipophorin III in Mormon crickets in the laboratory is
not in the free state. The insects were able to move
freely within the container, but it is not likely that
they moved as much as those in the field. They were
not observed struggling to escape, except when
handled.
We also found that total PO activity did not differ
among the dietary treatments for those Mormon
crickets released back into the band. However, total
PO activity was significantly affected by diet in the
Table 2 ANCOVA of log antibacterial activity following different
dietary treatments of captured-released Mormon crickets of differing gender and size
Factor
SS
F
P
Mass
0.044
d.f.
1
5.01
0.029
Sex
0.000
1
0.00
0.99
Diet
0.124
4
3.49
0.013
Model
0.166
6
3.13
0.0105
Error
0.479
54
captive animals. Those fed macronutrients, whether
P, C, or P þ C diets, in captivity had greater total PO
activity than those fed vitamins only. Spontaneous
PO activity was not affected by diet in Mormon
crickets released back into the band or in those
held captive. This result supports the conclusion
that carbohydrate-seeking bands are not limited in
PO activity (Srygley and Lorch 2011). It is interesting
that total PO activity, but not spontaneous PO activity, increased with body mass. In a previous study
of migrating Mormon crickets, spontaneous PO increased with body mass, and we hypothesized that
smaller individuals might be younger or malnourished (Srygley et al. 2009). From the day of eclosion
to adulthood, Mormon crickets under laboratory
conditions gain mass until at least 13 days old.
Spontaneous and total PO activities also increase
with age in laboratory-reared Mormon crickets
(Srygley 2012). Although gain in mass in the field
is likely to be more variable than in the laboratory,
the association between body mass and total PO is
probably due to an underlying association between
body mass and age.
Protein-limited Mormon crickets, such as those
found in migratory bands in Idaho (Simpson et al.
2006) and Utah (Srygley et al. 2009), are deficient
in spontaneous PO. When offered a protein-rich
diet, protein-limited crickets increase circulating PO
Fig. 4 Least-squares means of total PO activity (S.E.) covaried with body mass were not significantly dependent on diet in the releaserecapture experiment (open bars), whereas the effect of diet was significant in the laboratory experiment (gray bars; see legend of
Fig. 2 for abbreviations). Different letters above the columns indicate significant differences in multiple comparisons of the means
(P50.05). For comparison with the laboratory results, the dashed line indicates the mean for the Mormon crickets in the releaserecapture experiment.
1010
R. B. Srygley and P. D. Lorch
titers but did not elevate anti-bacterial activity.
Spontaneous PO titers are directly proportional to
the rate of encapsulation of foreign bodies introduced into the hemolymph (Srygley et al. 2009;
Srygley and Lorch 2011), and they are also associated
with attempted clearing of blastospores and hyphae
of the entomopathogenic fungus Beauveria bassiana
from the hemolymph (Srygley and Jaronski 2011).
In direct contrast, Mormon crickets that are carbohydrate-limited, such as those we found in
Nevada, increase anti-bacterial activity but not spontaneous PO activity following ingestion of carbohydrate-rich foods. Thus, we hypothesize that
nutritional deficiencies result in the development of
two types of migratory Mormon cricket, one that
shows a general reduction in immunocompetence,
making it more susceptible to pathogenic fungi,
and another that is susceptible to bacteria. The
Table 3 ANCOVA of total PO activity following dietary treatments of captured-released Mormon crickets of differing gender
and size
Factor
F
P
25,316,730
1
6.60
0.0122
Sex
2,504,258
1
0.65
0.42
Diet
31,030,382
4
2.02
0.0997
2.37
0.0373
Mass
SS
d.f.
Model
54,601,894
6
Error
291,543,658
76
challenge for the Mormon cricket is to balance
intake of carbohydrates and proteins in ways that
maximize resistance to both types of pathogen.
Effects of climate on macronutrient availability
Temperature and precipitation can have profound
effects on the availability of protein and carbohydrates (Jones and Coleman 1992). Studies of dietary
intake integrate behavioral decisions and physiological processes at the organismal level, demographic
processes at the population level, and patch dynamics of the metapopulation at the landscape level
(Simpson et al. 2010). However, in a simpler
system in which the Australian plague locust
Chortoicetes terminifera eats only two species of
grass, measures of protein and carbohydrate in the
host plants failed to predict assimilation or growth
(Clissold et al. 2006). The precise effects of abiotic
changes on the protein and carbohydrate sources
sought by the Mormon crickets have not been investigated, but we know that seeds, flower heads, and
invertebrates are protein-rich sources sought by
Mormon crickets and that fungi are a carbohydrate-rich source. In the water-limited rangeland of
the western United States, nitrogen limits net primary productivity and soil moisture increases the
ability of plants to absorb nitrogen from the soil
(Hooper and Johnson 1999; Yahdjian et al. 2011).
As a result, foliage becomes richer in protein in
wetter years, and the plants produce more proteinrich flowers and seeds. Rainfall not only influences
Fig. 5 Spontaneous PO activity (mean S.E.) for females (open bars) and males (gray bars) in the release-recapture experiment were
independent of diet (see legend of Fig. 2 for abbreviations).
1011
Diet deficiency, migration and immunity
Table 4 ANCOVA of log total PO activity of captive Mormon
crickets with blood drawn 4 or 24 h following dietary treatment
Factor
SS
Mass
0.0974
d.f.
1
F
P
7.76
0.0076
Sex
0.0140
1
1.11
0.30
Diet
0.1178
3
3.13
0.0340
Drawtime
0.0111
1
0.89
0.35
Model
0.2527
6
3.36
0.0075
Error
0.6151
49
Acknowledgments
Table 5 ANCOVA of PO activity of captive Mormon crickets
with blood drawn 4 or 24 h following dietary treatment
Factor
Mass
SS
d.f.
F
P
969
1
Sex
493473
1
Diet
43307
3
0.45
0.72
Drawtime
58911
1
1.82
0.18
3.52
0.0065
Model
683840
6
Error
1358134
42
to show increased susceptibility to bacteria. Few
studies have incorporated movement into investigations of resistance to disease (e.g., Adamo et al. 2008;
González-Tokman et al. 2011). Thus, differences in
diet and movement between insects in the laboratory
and those in nature call into question the validity of
applying the results of pathogen tests conducted in a
laboratory to field conditions.
0.03
15.3
We gratefully acknowledge Laura Senior (USDAARS) for assistance with fieldwork and with assays
conducted in the laboratory, and Neil Drinkard for
assistance in the field. We thank J. Gaskin and anonymous reviewers for critiquing earlier versions of this
manuscript.
0.86
0.0003
the amount of protein accessible to Mormon crickets
directly from plants but may also influence the abundance of high-protein, rangeland invertebrates upon
which Mormon crickets also forage. High density
bands of Mormon crickets will generally deplete
macronutrients locally and nutrients may become
limiting both in wet and in dry years. In drier habitats or years, we would expect Mormon crickets to
be protein-limited and less resistant to pathogenic
fungi, whereas in wetter habitats or years they
should be carbohydrate-limited and more susceptible
to bacterial pathogens. We are currently investigating
this hypothesized relationship between environment,
locomotion, and immunity in Mormon crickets.
This relationship between the environment, diet,
and immunity might apply to herbivorous insects in
general because they all share these innate defenses:
the PO cascade, including the proPO cascade, and
anti-bacterial enzymatic activity. Environmental
stress, such as moderate drought, can cause mobilization of nitrogen from plant tissues, thereby providing a better source of food for some herbivorous
insects (White 1984), but chewing and galling insects
generally perform less well on stressed plants
(Koricheva et al. 1998). Because we hypothesize
that anti-bacterial activity is compromised directly
by movement, carbohydrate-limited insects might
need to be dispersing or migrating to new habitats
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